O-polysaccharide is both the dominant immunogen and a major virulence determinant of Brucella spp. In the absence of O-polysaccharide classical Brucella species do not cause disease, and are rapidly cleared from :he host. Data from in vitro model systems appears less decisive. Although most evidence suggests rough mutants retain resistance to macrophage microbicidal killing mechanisms, some results suggest considerable replicative ability that may be masked by increased sensitivity to early killing mechanisms. However, there are also reports of cytotoxic cell death (CCD) induced by infection with rough mutants that is specific for "nacrophages. CCD is accompanied by elevated levels of tumor necrosis factor (TNFa), and nitric oxide (NO) and examination of macrophages revealed nuclear recruitment of nuclear factor kappa B (NF-kappaB) in macrophages exposed to rough organisms that remained in the cytoplasm of cells infected with smooth organisms. There is an increasing consensus that interaction between Brucella and the host cell may be in part controlled or altered by lipopolysaccharide (LPS) on the surface of Brucella. Our long-range goal is to identify the genes required for intracellular survival of Brucella. The objectives of !his application are to determine the contribution of O-polysaccharide in establishing a successful infection through its interaction with macrophages. Our central hypothesis is that O-polysaccharide is an essential element in the interaction between Brucella and host macrophages and enhances survival by evading or altering the innate immune response. The rationale for the proposed research is that understanding the interactions between smooth organisms and the host cell to elicit proper uptake and survival will enhance understanding of the mechanisms resulting in persistence and disease and provide information for the development of improved vaccines or treatments to enhance clearance of infections. We are particularly well prepared to study this interaction because we have established a Brucella mefitensis mutant bank and in vitro and in vivo models of infection and have the capacity to examine Brucella macrophage interaction at the cellular level and in animals models of infection using molecular, genomic and bioinformatic approaches.